Yersinia enterocolitica is one of three pathogenic species of the genus Yersinia. It is responsible for disease symptoms ranging from a mild gastroenteritis to systemic infection, which is often fatal. The symptoms and progression of infection are recapitulated in a mouse model, making this not only an important pathogen but also an excellent model system for studying virulence. A major component in the virulence of pathogenic Yersinia is a large plasmid that encodes a complete type three secretion system (the Ysc TTSS) including the apparatus, translocators, and effectors. Recently, the Ysa (Yersinia secretion apparatus) TTSS was discovered on the Y. enterocolitica chromosome and implicated as a virulence determinant. The ysa locus is not found in Y. pestis or Y. pseudotuberculosis, nor in non- or low-pathogenic Y. enterocolitica strains. The Ysa TTSS is most closely related to the SPI-1 TTSS of Salmonella and Mxi-Spa TTSS of Shigella. Thus, like Salmonella enterica, which has two TTSS that play different roles during the infectious process, the Ysc and Ysa TTSS may play distinct roles for Y. enterocolitica. Compared to the extensively studied Ysc system, little is known about the Ysa TTSS. At least eight Ysp's (Yersinia secreted proteins) are secreted through the Ysa TTSS, and in vitro secretion has only been observed at 26 degrees C in L-broth with high NaC1 concentrations. Proximal to the duster of genes believed to encode the apparatus are two genes with homology to a two-component regulatory system, YsrRS. Recent results from our laboratory indicated that YsrRS are important in regulating expression of ysa in response to high salt. The overall aims of this proposal are to better characterize the role of the Ysa TTSS in virulence and to better understand its regulation by YsrRS. The goals of our work are to test the hypotheses that (a) the Ysa TTSS plays a role in the early steps of infection, (b) YsrRS are important regulators of the Ysa system in response to NaC1 and possibly in response to other conditions, and (c) YsrRS regulate additional genes, some of which may play a role in virulence.